Summary of work: The transcription factor C/EBPalpha is a positive modulator of transcription for several adipocyte-specific genes which play a role in energy metabolism. However, there is little information available regarding regulation of its expression by metabolic signals. In a recent published paper, we reported that exposure to insulin for 5-24 h attenuated C/EBPalpha expression when 3T3-L1 adipocytes were incubated in 24 mM glucose, but not in 5.7 mM glucose. Nuclear run-on transcription assays indicated a transcriptional repression of C/EBPalpha gene, but not that of C/EBPbeta. Glucosamine, a product of the hexosamine pathway, mimicked the ability of high glucose to reduce C/EBPalpha expression in insulin-treated cells. Similar results were obtained with xylitol, an activator of the pentose phosphate pathway. There was no correlation between the accumulation of hexosamine pathway metabolites and/or changes in intracellular protein glycosylation with the ability of high glucose, glucosamine, or xylitol to down-regulate C/EBPalpha expression. None of these treatments caused a reduction in intracellular ATP levels. Stable transfection of 3T3-L1 cells with the 5-flanking 468-bp sequence of the mouse C/EBPalpha gene fused to luciferase demonstrated that promoter activity was also reduced by these nutrients. Of interest, treatment of rats with glucose or glucosamine led to a reduction in C/EBPalpha mRNA levels in epididymal, but not omental, fat. Taken together, these results suggest that metabolic signals serve to down-regulate C/EBPalpha expression both in vitro and in vivo. We plan to determine the signal transduction pathways used by these nutrients to control expression of the C/EBPalpha gene. Special emphasis will be given toward members of the mitogen-activated protein kinase family (ERK, p38 and JNK) and phosphatidylinositol 3-kinase. - Glucosamine; Signal transduction; Transcription factors; Cells in culture; Adipose tissue; Glucose metabolism.